Protein folding and misfolding represent a fundamental problem in biochemistry. How is the native state (the presumed thermodynamic ground state) reached on a physiologic timescale; in particular, how are misfolded states ("kinetic traps") avoided? What are the structures of protein-folding intermediates? Is there a "protein-folding code", and if so, is unique "foldability" a general property of protein sequences? These questions have long been of interest in physical biochemistry and biophysics. Recent advances in molecular medicine now highlight their central relevance to the pathogenesis of human diseases. We propose to dissect an ancestral protein-folding pathway: that of the insulin-IGF motif. Preliminary studies have established that oxidative folding of insulin-like growth factor 1 (IGF-1) yields two products (disulfide isomers) under thermodynamic control. Multidimensional NMR studies will be conducted of the IGF-1 isomer to determine its structural relationship to native IGF-1 and to a novel misfolded state of insulin (Nature Structural Biology 2, 129-138). Genetically engineered analogs and peptide models of protein-folding intermediates will be designed to test the following propositions:Hypothesis 1. That NMR studies of misfolded states (kinetic traps) will identify determinants of native protein folding; Hypothesis 2. That comparative studies of homologous folding pathways (proinsulin and IGF-1) will identify conserved intermediates and transition states; and Hypothesis 3. That the non-random pathway of disulfide bond formation will enable design of peptide and recombinant models of protein-folding intermediates. Of particular interest will be collaborative use of non-standard amino acids in solid-phase peptide synthesis to construct novel peptide models of protein-folding intermediates. By combining 2D and multidimensional NMR spectroscopy with modern methods of chemical synthesis and genetic engineering, this application offers the exciting possibility of delineating a pathway from protein sequence to structure.